Proposed investigations focus on fructose-1,6-bisphosphatase, an enzyme of gluconeogenesis subject to coordinate regulation by metabolites, and hexokinase isoforms I and II, enzymes that inhibit apoptosis through their association with mitochondria. A proposed model describes how small and localized ionformational change, induced by the binding of allosteric or active site inhibitors, triggers global conformation change in fructose-1,6-bisphosphatase. Directed mutations, the formation of hybrid tetramers by subunit exchange, fluorescence spectroscopy, kinetics, and structure determinations by x-ray diffraction, test the validity of the proposed model in accounting for positive cooperativity in allosteric inhibition, and iynergism between allosteric and active-site directed inhibitors. Research will define new sites from which an appropriate ligand can reinforce the action of physiological inhibitors of fructose-1,6-bisphosphatase, leading in the long run to new drugs that ameliorate high levels of serum glucose. The release of hexokinase isoforms I and II from mitochondria is a key step in apoptosis. Experiments here are designed to reveal the mechanism of release of hexokinase isoforms from the mitochondrion, and the subunit structure of hexokinase isoforms in their mitochondrion-bound states. Mitochondrial binding and release properties of mutant forms of hexokinase types I and II will test specifc mechanisms of ligand-induced release. Studies of spin-labeled hexokinase bound to reconsituted vesicles, using electron paramagnetic resonance methods, will determine whether the enzyme exists on the mitochondrion as a multimer, and if so the relative arrangement of subunits in that multimer. Investigations of fructose-1,6-bisphosphatase are of direct significance to the development of drugs that reduce high levels of serum glucose associated with diabetes type II. Work on hexokinase isoforms I and II may provide new strategies in triggering apoptosis (cell death) in cancers.